antibodies against lymphatic vessel hyaluronic receptor 1  (R&D Systems)

Journal: The Journal of Clinical Investigation

Article Title: Targeting lymphatic vessels enhances bone regeneration by augmenting osteoclast activity in mouse models of amputation

doi: 10.1172/JCI191906

Figure Lengend Snippet: ( A ) Schematic of lymphatic vessels in an unamputated digit, showing the normal ventral lymphatic network. ( B ) Immunofluorescence staining for lymphatic markers LYVE1 (shown in yellow) and PDPN (shown in magenta) in Prox1-eGFP reporter mice demonstrates lymphatic vessels localized to the ventral side of the unamputated digit. Scale bar: 200 μm. ( C ) Schematic showing lymphatic remodeling during the blastema stage 10 DPA. ( D ) Immunofluorescence at 10 DPA reveals expansion of lymphatic vessels toward the forming blastema. ( E ) Schematic of the early regenerative stage (21 DPA). ( F ) Immunofluorescence at 21 DPA shows persistent ventral lymphatics closely associated with regenerating tissue. ( G ) Schematic of late regeneration (35 DPA). ( H ) Immunofluorescence at 35 DPA demonstrates reestablishment of a ventral lymphatic architecture resembling the unamputated state. BM, bone marrow; DPA, days postamputation.

Article Snippet: For IF, frozen sections were washed in 1× TBS-T (0.05% Tween-20), blocked for 2 hours (1% BSA, 2% donkey/goat serum, 0.05% Triton X-100, 300 mM glycine, pH 8.4), and incubated overnight at 4°C with primary antibodies: LYVE1 (R&D Systems AF2125, 1:100), PDPN (Abcam ab11936, 1:100), CD45 (Cell Signaling Technology [CST] D3F8Q, 1:100), CD31 (CST 77699, 1:100), CD3 (Abcam ab16669, 1:100), and PROX1 (Abcam ab101851, 1:100).

Techniques: Immunofluorescence, Staining

Journal: The Journal of Clinical Investigation

Article Title: Targeting lymphatic vessels enhances bone regeneration by augmenting osteoclast activity in mouse models of amputation

doi: 10.1172/JCI191906

Figure Lengend Snippet: ( A ) Immunofluorescence staining for lymphatic markers PROX1 (green) and LYVE1 (red) in control and SAR131675-treated digits at 5 and 8 DPA. Scale bars, 100 μm. ( B ) Quantification of lymphatic vessel area (PROX1 + LYVE1 + ) shows significantly decreased lymphangiogenesis in SAR131675-treated digits at 8 DPA. Data are mean ± SD; Student’s t test; * P < 0.05. ( C ) Representative longitudinal micro-CT renderings of control (top) and SAR131675-treated (bottom) digits from 5 to 42 DPA show accelerated histolysis followed by enhanced bone regeneration in SAR131675-treated samples. ( D ) Quantification of bone volume over time demonstrates greater initial bone resorption and earlier regeneration in the SAR131675-treated group ( n = 4–12 digits per group; mean ± SD; * P < 0.05, ** P < 0.01). ( E – G ) Violin plots comparing ( E ) Flt4 (VEGFR3) expression, ( F ) LEC score, and ( G ) osteoclast gene score between control and SAR131675-treated digits. VEGFR3 inhibition reduced lymphatic gene expression while increasing osteoclast-associated signatures. ( H ) TRAP-stained sections of distal P3 bone at 5 and 8 DPA show markedly elevated osteoclast activity (pink) in SAR131675-treated digits compared with controls. ( I ) Quantification of erosion perimeter/bone perimeter (μm) reveals significantly increased bone erosion in SAR131675-treated digits at 5 and 8 DPA. ( J ) Osteoclast number per bone perimeter (μm) is significantly higher in the SAR131675-treated group at 8 DPA. Data are mean ± SD; Student’s t test; * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: For IF, frozen sections were washed in 1× TBS-T (0.05% Tween-20), blocked for 2 hours (1% BSA, 2% donkey/goat serum, 0.05% Triton X-100, 300 mM glycine, pH 8.4), and incubated overnight at 4°C with primary antibodies: LYVE1 (R&D Systems AF2125, 1:100), PDPN (Abcam ab11936, 1:100), CD45 (Cell Signaling Technology [CST] D3F8Q, 1:100), CD31 (CST 77699, 1:100), CD3 (Abcam ab16669, 1:100), and PROX1 (Abcam ab101851, 1:100).

Techniques: Immunofluorescence, Staining, Control, Micro-CT, Expressing, Inhibition, Gene Expression, Activity Assay

Journal: The Journal of Clinical Investigation

Article Title: Targeting lymphatic vessels enhances bone regeneration by augmenting osteoclast activity in mouse models of amputation

doi: 10.1172/JCI191906

Figure Lengend Snippet: ( A ) Proportional cell distribution derived from scRNA-Seq showing increased representation of macrophage and monocyte populations, while shows decrease in LEC population in SAR131675-treated samples at 5 DPA. ( B ) Immunofluorescent staining for CD45 (red) and LYVE1 (green) in control and SAR131675-treated digits at 5 DPA. Scale bar: 100 μm. ( C ) Quantification of CD45 + cells shows a significant increase in immune cell infiltration in VEGFR3-inhibited digits at 5 DPA (mean ± SD; *** P < 0.001; Student’s t test). ( D ) CyTOF analysis of immune cells from control and SAR131675-treated mice demonstrates expanded myeloid cell clusters (blue and orange arrows) under VEGFR3 inhibition. ( E and F ) Contour plots of F4/80 + cells show elevated frequencies of F4/80-high myeloid cells ( E ) and increased F4/80 + monocyte subsets ( F ) in SAR131675-treated mice compared with controls. ( G ) Contour plots of CD69 + CD44 + T cells in SAR131675-treated mice compared with controls. ( H ) Immunofluorescence staining for CD3 (green) and LYVE1 (red) at 5 and 8 DPA confirms elevated T cell infiltration in SAR131675-treated digits. Scale bar: 50 μm. ( I ) Quantification of CD3 + cells at 5 and 8 DPA shows a significant increase in the VEGFR3-inhibited group (* P < 0.05; mean ± SD; Student’s t test).

Article Snippet: For IF, frozen sections were washed in 1× TBS-T (0.05% Tween-20), blocked for 2 hours (1% BSA, 2% donkey/goat serum, 0.05% Triton X-100, 300 mM glycine, pH 8.4), and incubated overnight at 4°C with primary antibodies: LYVE1 (R&D Systems AF2125, 1:100), PDPN (Abcam ab11936, 1:100), CD45 (Cell Signaling Technology [CST] D3F8Q, 1:100), CD31 (CST 77699, 1:100), CD3 (Abcam ab16669, 1:100), and PROX1 (Abcam ab101851, 1:100).

Techniques: Derivative Assay, Staining, Control, Inhibition, Immunofluorescence

Journal: The Journal of Clinical Investigation

Article Title: Targeting lymphatic vessels enhances bone regeneration by augmenting osteoclast activity in mouse models of amputation

doi: 10.1172/JCI191906

Figure Lengend Snippet: ( A ) Immunofluorescent staining for lymphatic markers (PROX1, LYVE1, and PDPN) in control and Flt4Cre ERT2+ iDTR + mice shows fewer lymphatic vessels formed during regeneration in Flt4Cre ERT2+ iDTR + . ( B ) Quantification of lymphatic area within the tissue shows significant differences between the control and Flt4Cre ERT2+ iDTR + group at 5 DPA. ( C ) Representative micro-CT renderings of control digits (top) and a Flt4Cre ERT2+ iDTR + (bottom) 5, 8, 12, 16, and 28 DPA. ( D ) Quantification of bone volume at 5, 8, 12, 16, and 28 DPA. ( n = 4–12 digits/group.) ( E ) Histology images of amputated P3 bone stained with TRAP at 5 and 8 DPA comparing control and Flt4Cre ERT2+ iDTR + group. ( F ) Comparison of osteoclast-mediated bone erosion at 2 major osteolysis time points (5 and 8 DPA). ( G ) Comparison of osteoclast numbers at major osteolysis time point (8 DPA). Significance was determined using Student’s t test, with data presented as mean ± SD (* P < 0.05; *** P < 0.005; **** P < 0.0001).

Article Snippet: For IF, frozen sections were washed in 1× TBS-T (0.05% Tween-20), blocked for 2 hours (1% BSA, 2% donkey/goat serum, 0.05% Triton X-100, 300 mM glycine, pH 8.4), and incubated overnight at 4°C with primary antibodies: LYVE1 (R&D Systems AF2125, 1:100), PDPN (Abcam ab11936, 1:100), CD45 (Cell Signaling Technology [CST] D3F8Q, 1:100), CD31 (CST 77699, 1:100), CD3 (Abcam ab16669, 1:100), and PROX1 (Abcam ab101851, 1:100).

Techniques: Staining, Control, Micro-CT, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Targeting lymphatic vessels enhances bone regeneration by augmenting osteoclast activity in mouse models of amputation

doi: 10.1172/JCI191906

Figure Lengend Snippet: ( A ) Immunofluorescence staining for lymphatic markers (LYVE1 and PDPN) in sham and LNR digits at 8 DPA showing a marked reduction in lymphatic vessel density in LNR samples (yellow arrows). ( B ) Quantification of lymphatic area reveals a significant decrease in lymphatic coverage in LNR digits compared with sham controls ( P < 0.01; mean ± SD, Student’s t test). ( C ) Violin plot showing reduced lymphatic endothelial cell (LEC) score in LNR samples relative to sham controls ( P = 0.029). ( D ) Representative longitudinal micro-CT reconstructions of sham (top) and LNR (bottom) digits at 5, 8, 12, 16, 28, and 42 DPA. ( E ) Quantification of bone volume over time demonstrates accelerated bone regrowth in LNR digits (* P < 0.05, ** P < 0.01; n = 4–12 digits/group). ( F ) Violin plot showing elevated osteoclast scores in LNR digits ( P = 0.044). ( G ) Dot plot of osteoclast-related gene expression ( Mmp9 , Ctsk , Acp5 , Atp6v0d2 ) showing higher expression in LNR versus control samples. ( H ) TRAP staining of P3 bones at 3, 5, and 8 DPA illustrating increased osteoclast activity in LNR digits. ( I ) Quantification of osteoclast-mediated bone erosion at 5 and 8 DPA ( P < 0.05). ( J ) Quantification of osteoclast number at 8 DPA (** P < 0.001). Data are shown as mean ± SD, and significance was determined using Student’s t test.

Article Snippet: For IF, frozen sections were washed in 1× TBS-T (0.05% Tween-20), blocked for 2 hours (1% BSA, 2% donkey/goat serum, 0.05% Triton X-100, 300 mM glycine, pH 8.4), and incubated overnight at 4°C with primary antibodies: LYVE1 (R&D Systems AF2125, 1:100), PDPN (Abcam ab11936, 1:100), CD45 (Cell Signaling Technology [CST] D3F8Q, 1:100), CD31 (CST 77699, 1:100), CD3 (Abcam ab16669, 1:100), and PROX1 (Abcam ab101851, 1:100).

Techniques: Immunofluorescence, Staining, Micro-CT, Gene Expression, Expressing, Control, Activity Assay